Migraine is one of the most common neurovascular disorders. It is highly debilitating and difficult to treat. The disease mechanisms may involve abnormal ion homeostasis and neurotransmission. Multiple mutations in the gene encoding CaV2.1, the pore-forming subunit of human P/Q-type voltage-gated Ca2+ channel (VGCC), have been associated with familial hemiplegic migraine type 1 (FHM-1), a subclass of migraine with aura. Many of the migraine preventive and abortive drugs modulate the function of VGCCs but with poor specificity and efficacy. The overall goal of this project is to better understand th contribution of individual VGCC subtypes to the pathophysiology of migraine headache. In addition, the potential of VGCC blockers as anti-migraine therapeutics will be directly tested. Recent studies from our lab have found that loss-of-function (LOF) CaV2.1 mutations cause a decrease of P/Q- type current and a compensatory increase in N-type VGCC current in all subtypes of trigeminal ganglion (TG) and dorsal root ganglion neurons. Interestingly, a selective increase in LVA T-type VGCC current occurs only in small-diameter TG neurons that do not bind to isolectin B4 (IB4-). Moreover, LOF CaV2.1 mutations results in hyper-excitability of small IB4- neurons innervating the dura but not the facial skin. This is consistent with the fact that, other than migraine headache, FHM-1 patients do not show higher incidence of other somatic or orofacial pain. Importantly, pretreatment of wild-type mice with T- or N-type VGCC blockers significantly reduced the duration of head-directed nocifensive behavior in a mouse model of headache, indicating that VGCCs are involved in the activation of the neuronal circuit underlying migraine headache. In this project we propose to employ a multidisciplinary approach to investigate the mechanisms through which multiple VGCCs regulate the excitability and synaptic transmission of dural afferent neurons, thereby modulating the gain of the migraine circuit. First, we will test the hypothesis that P/Q-type Ca2+ channels regulate the excitability o small IB4- dural afferent neurons via functional coupling of the Ca2+-activated TRESK background K+ channels. We will investigate whether enhancing TRESK channel activity can reverse the hyper-excitability of dural afferent neurons and inhibit headache-like behavior in a mouse model. Secondly, we will use optical imaging technique to elucidate the contribution of N-type and other subtypes of VGCCs to Ca2+ influx synaptic transmission at dural afferent terminals. Finally, we will use a mouse model of headache to test the hypothesis that N- and T-type VGCCs in dural afferent neurons are potential targets for anti-migraine therapeutics. We will test whether N- and T-type blockers can be used for both abortive and preventive therapy of migraine headache. Taken together, the outcome of this project will offer new insights into the contribution of VGCCs to migraine pathophysiology as well as the therapeutic potential of trigeminal VGCCs and their downstream effector(s) in migraine treatment and prevention.